Auxin is the most important class of hormones in plants, and its asymmetric distribution pattern in plants regulates the developmental processes of plant cells and tissues, including cell division, cell elongation, landwardness, apical dominance, plant flowering and leaf order development, primary root development and lateral root genesis, senescence, and various stress responses.
The endogenous growth factors of plants include indole-3-acetic acid (IAA), 4-chloroindole-3-acetic acid (4-Cl-IAA), and phenylacetic acid (PAA). Among them, IAA is the most widely distributed and functional growth factor in plants.
The establishment of effective and practical methods for the qualitative and quantitative analysis of growth hormone and the real-time, accurate and in situ detection of growth hormone synthesis, distribution and transport in various tissues of plants is of great importance for the study of plant growth and development.
A series of new techniques and methods have been developed for the trace detection of auxin, including chromatography, mass spectrometry, fluorescence luminescence, biosensors and electrochemistry.
Immunoassay
IAA immunoassay is an immunological technique applied to the determination of IAA. Its basic principle is to use the specific competitive binding between antigen and antibody. IAA is used as the antigen to obtain the corresponding specific IAA antibody. A certain amount of labeled IAA molecules competes with an unknown amount of IAA molecules in the sample to bind to a certain amount of antibody, and then the amount of IAA in the sample is calculated.
IAA immunoassays are divided into 2 types: radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA). RIAs have high sensitivity and can detect ultra-trace amounts of growth factors, but require radioisotopes. Due to the instability of the isotope and the damage to the operator's body, RIAs are gradually being phased out. Enzyme-linked immunoassays are more safe, cheaper, and require less experimental space as well as equipment.
ELISA differs depending on its coupling enzyme protein. IAA in indoleacetic acid oxidase ELISA forms indole aldehyde under the action of IAA oxidase, which reduces the amount of IAA in the system. The remaining IAA interacts with FeCl3 in the presence of inorganic acid to form a red chelate. The magnitude of IAA oxidase activity can be expressed in terms of its rate of IAA destruction. While labeling the antibody with horseradish peroxidase, adding the corresponding reaction substrate to the reaction system, the reaction substrate is eventually converted from blue to yellow after a certain time of action. The IAA content of the sample can be determined by the shade of the color.
With more and more researchers using and improving the method, immunoassay has become a common method to locate the IAA content in plant tissues and organs.
Physical or chemical assay
Physical or chemical assays are mainly divided into two types: spectroscopic and chromatographic methods. The early detection of auxin used is the colorimetric method, but the sensitivity and specificity of the method is low, has been rarely used. Now the more commonly used methods are mainly ultraviolet absorption spectroscopy, infrared absorption spectroscopy and fluorescence method. Although they are simple, rapid and sensitive, they have low specificity and can only be used for general studies such as characterization and structure identification of molecules.
Chromatographic separation and integrated detection techniques are increasingly used in the detection of auxin. The principle of chromatographic separation is based on the difference in the retention properties of the sample in the chromatographic stationary phase and the mobile phase. After separation, a chromatographic outflow curve can be obtained based on the corresponding detection method. The time to peak (retention time) of a substance under certain chromatographic separation conditions is a fixed value and can therefore be used for qualitative purposes. The peak area is closely related to the content of the substance and can be used for quantification.
Endogenous plant growth hormones are extremely low and unstable in vitro, thus requiring high analytical sensitivity of detection methods. Gas chromatography and liquid chromatography have been rapidly developed with the addition of commercial columns and detectors and the introduction of new detection methods for the detection of almost all auxins due to their good separation and determination.
Chromatographic analysis alone cannot meet the requirements of qualitative and quantitative analysis of ultra-low content auxins. MS with HPLC, using isotopically labeled IAA as internal standard, can provide accurate quantitative determination of endogenous hormones in plant tissues based on the area ratio equal to the concentration ratio, which can give more detailed information on the structure of the substance and can better analyze the nature of sample components. Mass spectrometry coupled with chromatography has largely overcome the shortcomings of traditional chromatographic techniques in the qualitative and quantitative analysis of plant hormones.
Reference
- Kumar, R., Bohra, A., Pandey, A. K., Pandey, M. K., & Kumar, A. (2017). Metabolomics for plant improvement: status and prospects. Frontiers in plant science, 8, 1302.